Rna compositions comprising a buffer substance and methods for preparing, storing and using the same

Abstract

The present disclosure relates generally to the field of RNA compositions comprising a buffer substance, methods for preparing and storing such compositions, and the use of such compositions in therapy.

1 . A composition comprising (i) RNA; (ii) a cationically ionizable lipid; and (iii) an aqueous phase, wherein the aqueous phase comprises a buffer system comprising a buffer substance having the formula N(R 1 )(R 2 )(R 3 ), its N-oxide, or a protonated form thereof, wherein:
 each of R 1 , R 2 , and R 3  is independently selected from H, C 1-6  alkyl, C 1-6  alkylene-R 4 , CH(C 1-5  alkylene-R 4 ) 2 , and C(C 1-5  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is H, CH(C 1-5 alkylene-R 4 ) 2 , or C(C 1-5  alkylene-R 4 ) 3 ; or two of R 1 , R 2 , and R 3  join together with the nitrogen atom to form a 5- or 6-membered N-heterocyclic ring which is optionally substituted with one or two R 5 ;   each R 4  is independently selected from —OH, —O-(C 1-6  alkylene-OH), and —N(R 6 ) z (C 1-6  alkylene-OH) 2-z , wherein each z is independently selected from 0 and 1; and each R 6  is independently selected from H and C 1-3  alkyl; and   each R 5  is independently selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , CH(C 1-5  alkylene-R 4 ) 2 , and C(C 1-5  alkylene-R 4 ) 3 .   
     
     
         2 . The composition of  claim 1 , wherein each of R 1 , R 2 , and R 3  is independently selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , CH(C 1-5  alkylene-R 4 ) 2 , and C(C 1-5  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is CH(C 1-5  alkylene-R 4 ) 2  or C(C 1-5  alkylene-R 4 ) 3 , preferably each of R 1 , R 2 , and
 R 3  is independently selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , CH(C 1-3  alkylene-R 4 ) 2 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is CH(C 1-3  alkylene-R 4 ) 2  or C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-3  alkyl, C 1-3 alkylene-R 4 , CH(C 1-3  alkylene-R 4 ) 2 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is CH(C 1-3  alkylene-R 4 ) 2  or C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , CH(C 1-2  alkylene-R 4 ) 2 , and C(C 1-2 alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is CH(C 1-2  alkylene-R 4 ) 2  or C(C 1-2  alkylene-R 4 ) 3 .   
     
     
         3 . The composition of  claim 1 or 2 , wherein each of R 1 , R 2 , and R 3  is independently selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , and C(C 1-5  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is C(C 1-5  alkylene-R 4 ) 3 , preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-3  alkyl, C 1-3  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is C(C 1-3 alkylene-R 4 ) 3 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , and C(C 1-2  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is C(C 1-2 alkylene-R 4 ) 3 . 
     
     
         4 . The composition of any one of  claims 1 to 3 , wherein each of R 1 , R 2 , and R 3  is independently selected from C 1-6  alkyl and C 1-6  alkylene-R 4 , preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-4  alkyl and C 1-4  alkylene-R 4 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-3  alkyl and C 1-3  alkylene-R 4 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-2  alkyl and C 1-2  alkylene-R 4 . 
     
     
         5 . The composition of any one of  claims 1 to 4 , wherein each R 4  is independently selected from —OH, —O-(C 1-4  alkylene-OH), and —N(R 6 ) z -(C 1-4  alkylene-OH) 2-z , wherein each z is independently selected from 0 and 1; and each R 6  is independently selected from H and C 1-3  alkyl, preferably each R 4  is independently selected from —OH, —O-(C 1-3  alkylene-OH), and —N(R 6 ) z -(C 1-3  alkylene-OH) 2-z , wherein each z is independently selected from 0 and 1; and each R 6  is independently selected from H and C 1-3  alkyl, more preferably each R 4  is independently selected from —OH, —O-(C 1-2  alkylene-OH), and —N(R 6 ) z -(C 1-2  alkylene-OH) 2-z , wherein each z is independently selected from 0 and 1; and each R 6  is independently selected from H and C 1-2  alkyl. 
     
     
         6 . The composition of any one of  claims 1 to 5 , wherein each R 4  is independently selected from —OH, —O-(C 1-4  alkylene-OH), and —N(C 1-4  alkylene-OH) 2 , preferably each R 4  is independently selected from —OH, —O-(C 1-3  alkylene-OH), and —N(C 1-3  alkylene-OH) 2 , more preferably each R 4  is independently selected from —OH, —O-(C 1-2  alkylene-OH), and —N(C 1-2  alkylene-OH) 2 . 
     
     
         7 . The composition of any one of  claims 1 to 6 , wherein each R 4  is independently selected from —OH, 2-hydroxyethoxy, and bis(2-hydroxyethyl)amino. 
     
     
         8 . The composition of any one of  claims 1 to 7 , wherein each of R 1 , R 2 , and R 3  is independently selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, 2-[bis(2-hydroxyethyl)amino]ethyl, and 1,5-dihydroxy-3-(2-hydroxyethyl)pentan-3-yl. 
     
     
         9 . The composition of any one of  claims 1 to 8 , wherein all of R 1 , R 2 , and R 3  are the same. 
     
     
         10 . The composition of  claim 9 , wherein all of R 1 , R 2 , and R 3  are methyl, ethyl, or 2-hydroxyethyl. 
     
     
         11 . The composition of any one of  claims 1 to 8 , wherein R 1  and R 2  are the same and R 3  differs from R 1  and R 2 . 
     
     
         12 . The composition of  claim 11 , wherein each of R 1  and R 2  is 2-hydroxyethyl, ethyl, or methyl. 
     
     
         13 . The composition of  claim 11 or 12 , wherein R 3  is selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, 2-[bis(2-hydroxyethyl)amino]ethyl, and 1,5-dihydroxy-3-(2-hydroxyethyl)pentan-3-yl. 
     
     
         14 . The composition of  claim 1 , wherein R 1  and R 2  join together with the nitrogen atom to form a 5- or 6-membered N-heterocyclic ring which is optionally substituted with one or two R 5 . 
     
     
         15 . The composition of  claim 14 , wherein R 3  is selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , and C(C 1-5  alkylene-R 4 ) 3 , preferably R 3  is selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 3  is selected from C 1-3  alkyl, C 1-2  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 3  is selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , and C(C 1-2  alkylene-R 4 ) 3 , more preferably R 3  is selected from selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, and 2-[bis(2-hydroxyethyl)amino]ethyl. 
     
     
         16 . The composition of  claim 14 or 15 , wherein the N-heterocyclic ring is a monocyclic ring containing at least one nitrogen ring atom and optionally one further ring heteroatom selected from O and S. 
     
     
         17 . The composition of any one of  claims 14 to 16 , wherein the N-heterocyclic ring is a monocyclic ring containing (i) one nitrogen ring atom; (ii) two nitrogen ring atoms; (iii) one nitrogen ring atom and one oxygen ring atom; (iv) one nitrogen ring atom and one sulfur ring atom; or (v) three nitrogen ring atoms. 
     
     
         18 . The composition of any one of  claim 14 to 17 , wherein the N-heterocyclic ring is a monocyclic 5- or 6-membered N-heterocyclic ring, such as is a monocyclic 6-membered N-heterocyclic ring. 
     
     
         19 . The composition of any one of  claims 14 to 18 , wherein the N-heterocyclic ring is selected from pyrrolidinyl, imidazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, 1,2-diazinanyl, 1,3-diazinanyl, 1,3,5-triazinanyl, morpholinyl, and thiomorpholinyl, preferably selected from piperidinyl, piperazinyl, 1,2-diazinanyl, 1,3-diazinanyl, morpholinyl, and thiomorpholinyl. 
     
     
         20 . The composition of any one of  claims 14 to 19 , wherein, if the N-heterocyclic ring contains only one nitrogen ring atom, this nitrogen ring atom is substituted with R 3 , R 3  being other than H, or, if the N-heterocyclic ring contains more than one nitrogen ring atom, one nitrogen ring atom is substituted with R 3 , R 3  being other than H, and at least one of the other nitrogen ring atoms, preferably each of the other nitrogen ring atoms, is substituted with R 5 . 
     
     
         21 . The composition of any one of  claims 14 to 20 , wherein each R 5  is independently selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , and C(C 1-5  alkylene-R 4 ) 3 , preferably R is selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 5  is selected from C 1-3  alkyl, C 1-3  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 5  is selected from C 1-2  alkyl, C 1-2 alkylene-R 4 , and C(C 1-2  alkylene-R 4 ) 3 , more preferably R is selected from selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, and 2-[bis(2-hydroxyethyl)amino]ethyl. 
     
     
         22 . The composition of any one of  claims 14 to 21 , wherein the N-heterocyclic ring is piperidinyl and the ring N atom is substituted with R 3 , R 3  being other than H. 
     
     
         23 . The composition of any one of  claims 14 to 21 , wherein the N-heterocyclic ring is piperazinyl, one the two ring N atoms is substituted with R 3 , R 3  being other than H, and the other ring N atom is optionally substituted with R 5 , preferably the other ring N atom is substituted with R 5 . 
     
     
         24 . The composition of  claim 23 , wherein both ring N atoms are substituted and R 5  is selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , and C(C 1-5  alkylene-R 4 ) 3 , preferably R 5  is selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 5  is selected from C 1-3  alkyl, C 1-3 alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 5  is selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , and C(C 1-2  alkylene-R 4 ) 3 , more preferably R 5  is selected from selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, and 2-[bis(2-hydroxyethyl)amino]ethyl. 
     
     
         25 . The composition of any one of  claims 14 to 24 , wherein each R 4  is independently selected from —OH, —O-(C 1-4  alkylene-OH), and —N(C 1-4  alkylene-OH) 2 , preferably each R 4  is independently selected from —OH, —O-(C 1-3  alkylene-OH), and —N(C 1-3  alkylene-OH) 2 , more preferably each R 4  is independently selected from —OH, —O-(C 1-2  alkylene-OH), and —N(C 1-2  alkylene-OH) 2 . 
     
     
         26 . The composition of any one of  claims 14 to 25 , wherein each R 4  is independently selected from —OH, 2-hydroxyethoxy, and bis(2-hydroxyethyl)amino. 
     
     
         27 . The composition of any one of  claims 14 to 26 , wherein R 3  and R 5  are the same. 
     
     
         28 . The composition of  claim 27 , wherein both of R 3  and R 1  are methyl, ethyl, 2-hydroxyethyl, or 2-(2-hydroxyethoxy)ethyl, preferably, both of R 3  and R 5  are 2-hydroxyethyl. 
     
     
         29 . The composition of any one of  claims 14 to 26 , wherein R 3  and R 5  differ from each other. 
     
     
         30 . The composition of  claim 1 , wherein R 1  is H. 
     
     
         31 . The composition of  claim 30 , wherein each of R 2  and R 3  is independently selected from C 1-6 alkyl, C 1-6  alkylene-R 4 , CH(C 1-5  alkylene-R 4 ) 2 , and C(C 1-5  alkylene-R 4 ) 3 , wherein at most one of R 2  and R 3  is CH(C 1-5  alkylene-R 4 ) 2  or C(C 1-5  alkylene-R 4 ) 3 , preferably each of R 2  and R 3  is independently selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , CH(C 1-3  alkylene-R 4 ) 2 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 2  and R 3  is CH(C 1-3  alkylene-R 4 ) 2  or C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 2  and R 3  is independently selected from C 1-3  alkyl, C 1-3  alkylene-R 4 , CH(C 1-3  alkylene-R 4 ) 2 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 2  and R 3  is CH(C 1-3  alkylene-R 4 ) 2  or C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 2  and R 3  is independently selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , CH(C 1-2  alkylene-R 4 ) 2 , and C(C 1-2  alkylene-R 4 ) 3 , wherein at most one of R 2  and R 3  is CH(C 1-2  alkylene-R 4 ) 2  or C(C 1-2  alkylene-R 4 ) 3 . 
     
     
         32 . The composition of  claim 30 or 31 , wherein each of R 2  and R 3  is independently selected from C 1-6  alkyl, C 1-6 alkylene-R 4 , and C(C 1-5  alkylene-R 4 ) 3 , wherein at most one of R 2  and R 3  is C(C 1-5  alkylene-R 4 ) 3 , preferably each of R 2  and R 3  is independently selected from C 1-4  alkyl, C 1-4 alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 2  and R 3  is C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 2  and R 3  is independently selected from C 1-3  alkyl, C 1-3  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 2  and R 3  is C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 2  and R 3  is independently selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , and C(C 1-2  alkylene-R 4 ) 3 , wherein at most one of R 2  and R 3  is C(C 1-2  alkylene-R 4 ) 3 . 
     
     
         33 . The composition of any one of  claims 30 to 32 , wherein each of R 2  and R 3  is independently selected from C 1-6  alkyl and C 1-6  alkylene-R 4 , preferably each of R 2  and R 3  is independently selected from C 1-4  alkyl and C 1-4  alkylene-R 4 , more preferably each of R 2  and R 3  is independently selected from C 1-3  alkyl and C 1-3  alkylene-R 4 , more preferably each of R 2  and R 3  is independently selected from C 1-2  alkyl and C 1-2  alkylene-R 4 . 
     
     
         34 . The composition of any one of  claims 30 to 33 , wherein each R 4  is independently selected from —OH, —O-(C 1-4  alkylene-OH), and —N(C 1-4  alkylene-OH) 2 , preferably each R 4  is independently selected from —OH, —O-(C 1-3  alkylene-OH), and —N(C 1-3  alkylene-OH) 2 , more preferably each R 4  is independently selected from —OH, —O-(C 1-2  alkylene-OH), and —N(C 1-2  alkylene-OH) 2 . 
     
     
         35 . The composition of any one of  claims 30 to 34 , wherein each R 4  is independently selected from —OH, 2-hydroxyethoxy, and bis(2-hydroxyethyl)amino. 
     
     
         36 . The composition of any one of  claims 30 to 35 , wherein each of R 2  and R 3  is independently selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, 2-[bis(2-hydroxyethyl)amino]ethyl, and 1,5-dihydroxy-3-(2-hydroxyethyl)pentan-3-yl, preferably, both of R 2  and R 3  are 2-hydroxyethyl or 2-(2-hydroxyethoxy)ethyl. 
     
     
         37 . The composition of any one of  claims 1 to 36 , wherein the buffer substance is selected from bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris-methane or BTM) and its protonated form, triethanolamine (TEA) and its protonated form, ethyldiethanolamine and its protonated form, 2-(diethylamino)ethan-1-ol and its protonated form, triethylamine and its protonated form, 2-[2-(diethylamino)ethoxy]ethan-1-ol and its protonated form, diethanolamine and its protonated form, N,N′-bis(2-hydroxyethyl)piperazine and its protonated form, N,N,N′,N′-tetrakis(2-hydroxyethyl)ethylenediamine and its protonated form, and trimethylamine N-oxide and its protonated form. 
     
     
         38 . The composition of any one of  claims 1 to 37 , wherein the buffer substance comprises at least one C 1-6  alkylene-R 4  (such as 2-hydroxyethyl) moiety. 
     
     
         39 . A composition comprising (i) RNA; and (ii) an aqueous phase, wherein the aqueous phase comprises a buffer system comprising a buffer substance having the formula N(R 1 )(R 2 )(R 3 ), its N-oxide, or a protonated form thereof, wherein:
 each of R 1 , R 2 , and R 3  is independently selected from H, C 1-6  alkyl, C 1-6  alkylene-R 4 , CH(C 1-5  alkylene-R 4 ) 2 , and C(C 1-5  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is H, CH(C 1-5  alkylene-R 4 ) 2 , or C(C 1-5  alkylene-R 4 ) 3 ; or two of R 1 , R 2 , and R 3  join together with the nitrogen atom to form a 5- or 6-membered N-heterocyclic ring which is optionally substituted with one or two R 5 ;   each R 4  is independently selected from —OH, —O-(C 1-6  alkylene-OH), and —N(R 6 ) z -(C 1-6  alkylene-OH) 2-z , wherein each z is independently selected from 0 and 1; and each R 6  is independently selected from H and C 1-3  alkyl; and   each R 5  is independently selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , CH(C 1-5  alkylene-R 4 ) 2 , and C(C 1-5  alkylene-R 4 ) 3 .   
     
     
         40 . The composition of  claim 39 , wherein each of R 1 , R 2 , and R 3  is independently selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , CH(C 1-5  alkylene-R 4 ) 2 , and C(C 1-5  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is CH(C 1-5  alkylene-R 4 ) 2  or C(C 1-5  alkylene-R 4 ) 3 , preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , CH(C 1-3  alkylene-R 4 ) 2 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is CH(C 1-3  alkylene-R 4 ) 2  or C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-3  alkyl, C 1-3 alkylene-R 4 , CH(C 1-3  alkylene-R 4 ) 2 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is CH(C 1-3  alkylene-R 4 ) 2  or C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , CH(C 1-2  alkylene-R 4 ) 2 , and C(C 1-2 alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is CH(C 1-2  alkylene-R 4 ) 2  or C(C 1-2  alkylene-R 4 ) 3 . 
     
     
         41 . The composition of  claim 39 or 40 , wherein each of R 1 , R 2 , and R 3  is independently selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , and C(C 1-5  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is C(C 1-5  alkylene-R 4 ) 3 , preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-3  alkyl, C 1-3  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is C(C 1-3  alkylene-R 4 ) 3 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , and C(C 1-2  alkylene-R 4 ) 3 , wherein at most one of R 1 , R 2 , and R 3  is C(C 1-2 alkylene-R 4 ) 3 . 
     
     
         42 . The composition of any one of  claims 39 to 41 , wherein each of R 1 , R 2 , and R 3  is independently selected from C 1-6  alkyl and C 1-6  alkylene-R 4 , preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-4  alkyl and C 1-4  alkylene-R 4 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-3  alkyl and C 1-3  alkylene-R 4 , more preferably each of R 1 , R 2 , and R 3  is independently selected from C 1-2  alkyl and C 1-2  alkylene-R 4 . 
     
     
         43 . The composition of any one of  claims 39 to 42 , wherein each R 4  is independently selected from —OH, —O-(C 1-4  alkylene-OH), and —N(R 6 ) z -(C 1-4  alkylene-OH) 2-z , wherein each z is independently selected from 0 and 1; and each R 6  is independently selected from H and C 1-3  alkyl, preferably each R 4  is independently selected from —OH, —O-(C 1-3  alkylene-OH), and —N(R 6 ) z -(C 1-3  alkylene-OH) 2-z , wherein each z is independently selected from 0 and 1; and each R 6  is independently selected from H and C 1-3  alkyl, more preferably each R 4  is independently selected from —OH, —O-(C 1-2  alkylene-OH), and —N(R 6 ) z -(C 1-2  alkylene-OH) 2-z , wherein each z is independently selected from 0 and 1; and each R 6  is independently selected from H and C 1-2  alkyl. 
     
     
         44 . The composition of any one of  claims 39 to 43 , wherein each R 4  is independently selected from —OH, —O-(C 1-4  alkylene-OH), and —N(C 1-4  alkylene-OH) 2 , preferably each R 4  is independently selected from —OH, —O-(C 1-3  alkylene-OH), and —N(C 1-3  alkylene-OH) 2 , more preferably each R 4  is independently selected from —OH, —O-(C 1-2  alkylene-OH), and —N(C 1-2  alkylene-OH) 2 . 
     
     
         45 . The composition of any one of  claims 39 to 44 , wherein each R 4  is independently selected from —OH, 2-hydroxyethoxy, and bis(2-hydroxyethyl)amino. 
     
     
         46 . The composition of any one of  claims 39 to 45 , wherein each of R 1 , R 2 , and R 3  is independently selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, 2-[bis(2-hydroxyethyl)amino]ethyl, and 1,5-dihydroxy-3-(2-hydroxyethyl)pentan-3-yl. 
     
     
         47 . The composition of any one of  claims 39 to 46 , wherein all of R 1 , R 2 , and R 3  are the same. 
     
     
         48 . The composition of  claim 47 , wherein all of R 1 , R 2 , and R 3  are methyl, ethyl, or 2-hydroxyethyl. 
     
     
         49 . The composition of any one of  claims 39 to 46 , wherein R 1  and R 2  are the same and R 3  differs from R 1  and R 2 . 
     
     
         50 . The composition of  claim 49 , wherein each of R 1  and R 2  is 2-hydroxyethyl, ethyl, or methyl. 
     
     
         51 . The composition of  claim 49 or 50 , wherein R 3  is selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, 2-[bis(2-hydroxyethyl)amino]ethyl, and 1,5-dihydroxy-3-(2-hydroxyethyl)pentan-3-yl. 
     
     
         52 . The composition of  claim 39 , wherein R 1  and R 2  join together with the nitrogen atom to form a 5- or 6-membered N-heterocyclic ring which is optionally substituted with one or two R 5 . 
     
     
         53 . The composition of  claim 52 , wherein R 3  is selected from C 1-6  alkyl, C 1 b alkylene-R 4 , and C(C 1-5 alkylene-R 4 ) 3 , preferably R 3  is selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 3  is selected from C 1-3  alkyl, C 1 a alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 3  is selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , and C(C 1-2  alkylene-R 4 ) 3 , more preferably R 3  is selected from selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, and 2-[bis(2-hydroxyethyl)amino]ethyl. 
     
     
         54 . The composition of any one of  claims 52 to 53 , wherein, if the N-heterocyclic ring contains only one nitrogen ring atom, this nitrogen ring atom is substituted with R 3 , R 3  being other than H, or, if the N-heterocyclic ring contains more than one nitrogen ring atom, one nitrogen ring atom is substituted with R 3 , R 3  being other than H, and at least one of the other nitrogen ring atoms, preferably each of the other nitrogen ring atoms, is substituted with R 5 . 
     
     
         55 . The composition of any one of  claims 52 to 54 , wherein each R 5  is independently selected from C 1-6  alkyl, C 1-6  alkylene-R 4 , and C(C 1-5  alkylene-R 4 ) 3 , preferably R 5  is selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 5  is selected from C 1-3  alkyl, C 1-3 alkylene-R 4 , and C(C 1-5  alkylene-R 4 ) 3 , more preferably R 5  is selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , and C(C 1-2  alkylene-R 4 ) 3 , more preferably R 5  is selected from selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, and 2-[bis(2-hydroxyethyl)amino]ethyl. 
     
     
         56 . The composition of any one of  claims 52 to 55 , wherein the N-heterocyclic ring is piperidinyl and the ring N atom is substituted with R 3 , wherein R 3  is other than H. 
     
     
         57 . The composition of any one of  claims 52 to 55 , wherein the N-heterocyclic ring is piperazinyl, one the two ring N atoms is substituted with R 3 , R 3  being other than H, and the other ring N atom is optionally substituted with R 5 , preferably the other ring N atom is substituted with R 5 . 
     
     
         58 . The composition of  claim 57 , wherein both ring N atoms are substituted and R 5  is selected from C 1-6  alkyl, C 1 (alkylene-R 4 , and C(C 1-5  alkylene-R 4 ) 3 , preferably R 5  is selected from C 1-4  alkyl, C 1-4  alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 5  is selected from C 1-3  alkyl, C 1-3 alkylene-R 4 , and C(C 1-3  alkylene-R 4 ) 3 , more preferably R 5  is selected from C 1-2  alkyl, C 1-2  alkylene-R 4 , and C(C 1-2  alkylene-R 4 ) 3 , more preferably R 5  is selected from selected from methyl, ethyl, 2-hydroxyethyl, 2-(2-hydroxyethoxy)ethyl, and 2-[bis(2-hydroxyethyl)amino]ethyl. 
     
     
         59 . The composition of any one of  claims 52 to 58 , wherein each R 4  is independently selected from —OH, —O-(C 1-4  alkylene-OH), and —N(C 1-4  alkylene-OH) 2 , preferably each R 4  is independently selected from —OH, —O-(C 1-3  alkylene-OH), and —N(C 1-3  alkylene-OH) 2 , more preferably each R 4  is independently selected from —OH, —O-(C 1-2  alkylene-OH), and —N(C 1-2  alkylene-OH) 2 . 
     
     
         60 . The composition of any one of  claims 52 to 59 , wherein each R 4  is independently selected from —OH, 2-hydroxyethoxy, and bis(2-hydroxyethyl)amino. 
     
     
         61 . The composition of any one of  claims 52 to 60 , wherein R 3  and R 5  are the same. 
     
     
         62 . The composition of  claim 61 , wherein both of R 3  and R 5  are methyl, ethyl, 2-hydroxyethyl, or 2-(2-hydroxyethoxy)ethyl, preferably, both of R 3  and R 5  are 2-hydroxyethyl. 
     
     
         63 . The composition of any one of  claims 52 to 60 , wherein R 3  and R 5  differ from each other. 
     
     
         64 . The composition of any one of  claims 39 to 63 , wherein the buffer substance is selected from bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris-methane or BTM) and its protonated form, triethanolamine (TEA) and its protonated form, ethyldiethanolamine and its protonated form, 2-(diethylamino)ethan-1-ol and its protonated form, triethylamine and its protonated form, 2-[2-(diethylamino)ethoxy]ethan-1-ol and its protonated form, diethanolamine and its protonated form, N,N′-bis(2-hydroxyethyl)piperazine and its protonated form, N,N,N′,N′-tetrakis(2-hydroxyethyl)ethylenediamine and its protonated form, and trimethylamine N-oxide and its protonated form. 
     
     
         65 . The composition of any one of  claims 39 to 64 , wherein the buffer substance comprises at least one C 1-6  alkylene-R 4  (such as 2-hydroxyethyl) moiety. 
     
     
         66 . The composition of any one of  claims 1 to 65 , wherein the buffer system further comprises an anion selected from the group consisting of chloride, acetate, glycolate, lactate, and the anion of a di- or tricarboxylic acid, such as the anion of citric acid, succinic acid, malonic acid, glutaric acid, or adipic acid. 
     
     
         67 . The composition of any one of  claims 1 to 66 , wherein the concentration of the buffer substance in the composition is between about 10 mM and about 200 mM, preferably between about 15 mM and about 100 mM, more preferably between about 20 mM and about 80 mM, more preferably between about 40 mM and about 60 mM, such as about 50 mM. 
     
     
         68 . The composition of any one of  claims 1 to 67 , wherein the pH of the composition is between about 4.0 and about 8.0, preferably between about 4.5 and about 8.0, such as between about 5.0 and about 8.0, between about 5.5 and about 8.0, between about 6.0 and about 8.0, between about 6.5 and about 8.0, between about 6.8 and about 7.9, or between about 7.0 and about 7.8. 
     
     
         69 . The composition of any one of  claims 1 to 68 , wherein water is the main component in the composition and/or the total amount of solvent(s) other than water contained in the composition is less than about 0.5% (v/v). 
     
     
         70 . The composition of any one of  claims 1 to 69 , wherein the osmolality of the composition is at most about 1000×10 −3  osmol/kg, preferably between about 100×10 −3  osmol/kg and about 750×10 −3  osmol/kg, such as between about 100×10 −3  osmol/kg and about 500×10 −3  osmol/kg, more preferably about 300×10 −3  osmol/kg. 
     
     
         71 . The composition of any one of  claims 1 to 70 , wherein the concentration of the RNA in the composition is about 5 mg/l to about 500 mg/l, such as about 10 mg/l to about 400 mg/l, about 10 mg/l to about 300 mg/l, about 10 mg/l to about 200 mg/l, about 10 mg/l to about 150 mg/1, or about 10 mg/l to about 100 mg/l, preferably about 10 mg/l to about 140 mg/l, more preferably about 20 mg/l to about 130 mg/l, more preferably about 30 mg/l to about 120 mg/l. 
     
     
         72 . The composition of any one of  claims 1 to 71 , wherein the composition comprises a cryoprotectant. 
     
     
         73 . The composition of any one of  claims 1 to 38 and 66 to 72 , wherein the cationically ionizable lipid comprises a head group which includes at least one nitrogen atom which is capable of being protonated under physiological conditions. 
     
     
         74 . The composition of any one of  claims 1 to 38 and 66 to 73 , wherein the cationically ionizable lipid has the structure of Formula (X) 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: 
         one of L 10  and L 20  is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) x —, —S—S—, —C(═O)S—, SC(═O)—, —NR a C(═O)—, —C(═O)NR a —, NR a C(═O)NR a —, —OC(═O)NR a — or —NR a C(═O)O—, and the other of L 10  and L 20  is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) x —, —S—S—, —C(═O)S—, SC(═O)—, —NR a C(═O)—, —C(═O)NR a —, NR a C(═O)NR a —, —OC(═O)NR a — or —NR a C(═O)O— or a direct bond; 
         G 1  and G 2  are each independently unsubstituted C 1 -C 12  alkylene or C 2-12  alkenylene; 
         G 3  is C 1-24  alkylene, C 2-24  alkenylene, C 3-8  cycloalkylene, or C 3-8  cycloalkenylene; 
         R a  is H or C 1-12  alkyl; 
         R 35  and R 36  are each independently C 6-24  alkyl or C 6-24  alkenyl; 
         R 37  is H, OR 50 , CN, —C(═O)OR 40 , —OC(═O)R 40  or —NR 50 C(═O)R 40 ; 
         R 40  is C 1-12  alkyl; 
         R 50  is H or C 1-6  alkyl; and 
         x is 0, 1 or 2. 
       
     
     
         75 . The composition of any one of  claims 1 to 38 and 66 to 73 , wherein the cationic or cationically ionizable lipid has the structure of Formula (XI): 
       
         
           
           
               
               
           
         
         wherein 
         each of R 1  and R 2  is independently R 5  or -G 1 -L 1 -R 6 , wherein at least one of R 1  and R 2  is -G 1 -L 1 -R 6 ; 
         each of R 3  and R 4  is independently selected from the group consisting of C 1-6  alkyl, C 2-6  alkenyl, aryl, and C 3-10  cycloalkyl; 
         each of R 5  and R 6  is independently a non-cyclic hydrocarbyl group having at least 10 carbon atoms; 
         each of G 1  and G 2  is independently unsubstituted C 1-12  alkylene or C 2-12  alkenylene; 
         each of L 1  and L 2  is independently selected from the group consisting of —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) x —, —S—S—, —C(═O)S—, —SC(═O)—, —NR a C(═O)—, —C(═O)NR a —, —NR a C(═O)NR a —, —OC(═O)NR a — and —NR a C(═O)O—; 
         Ra is H or C 1-12  alkyl; 
         m is 0, 1, 2, 3, or 4; and 
         x is 0, 1 or 2. 
       
     
     
         76 . The composition of any one of  claims 1 to 38 and 66 to 75 , wherein the cationically ionizable lipid comprises from about 20 mol % to about 80 mol %, preferably from about 25 mol % to about 65 mol %, more preferably from about 30 mol % to about 50 mol %, such as from about 40 mol % to about 50 mol %, of the total lipid present in the composition. 
     
     
         77 . The composition of any one of  claims 1 to 76 , which further comprises one or more additional lipids, preferably selected from the group consisting of polymer conjugated lipids, neutral lipids, steroids, and combinations thereof, more preferably the composition comprises the cationically ionizable lipid, a polymer conjugated lipid, a neutral lipid (e.g., a phospholipid), and a steroid. 
     
     
         78 . The composition of  claim 77 , wherein the polymer conjugated lipid comprises a pegylated lipid, wherein the pegylated lipid preferably (i) is selected from the group consisting of DSPE-PEG, DOPE-PEG, DPPE-PEG, and DMPE-PEG; or (ii) has the following structure: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: 
         R 12  and R 13  are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. 
       
     
     
         79 . The composition of  claim 77 , wherein the polymer conjugated lipid comprises a polysarcosine-lipid conjugate or a conjugate of polysarcosine and a lipid-like material, wherein the polysarcosine-lipid conjugate or conjugate of polysarcosine and a lipid-like material preferably is a member selected from the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate, and a mixture thereof. 
     
     
         80 . The composition of any one of  claims 77 to 79 , wherein the polymer conjugated lipid comprises from about 0.5 mol % to about 5 mol %, preferably from about 1 mol % to about 5 mol %, more preferably from about 1 mol % to about 4.5 mol % of the total lipid present in the composition. 
     
     
         81 . The composition of any one of  claims 77 to 80 , wherein the neutral lipid is a phospholipid, preferably selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl-phosphatidylethanolamine (DPyPE). 
     
     
         82 . The composition of any one of  claims 77 to 81 , wherein the neutral lipid comprises from about 5 mol % to about 40 mol %, preferably from about 5 mol % to about 20 mol %, more preferably from about 5 mol % to about 15 mol % of the total lipid present in the composition. 
     
     
         83 . The composition of any one of  claims 77 to 82 , wherein the steroid comprises a sterol such as cholesterol. 
     
     
         84 . The composition of any one of  claims 77 to 83 , wherein the steroid comprises from about 10 mol % to about 65 mol %, preferably from about 20 mol % to about 60 mol %, more preferably from about 30 mol % to about 50 mol % of the total lipid present in the composition. 
     
     
         85 . The composition of any one of  claims 77 to 84 , which comprises a cationically ionizable lipid, a polymer conjugated lipid, a neutral lipid (e.g., a phospholipid), and a steroid, wherein the cationically ionizable lipid comprises from about 30 mol % to about 50 mol %, such as from about 40 mol % to about 50 mol %, of the total lipid present in the composition; the polymer conjugated lipid comprises from about 1 mol % to about 4.5 mol % of the total lipid present in the composition; the neutral lipid (e.g., phospholipid) comprises from about 5 mol % to about 15 mol % of the total lipid present in the composition; and the steroid comprises from about 30 mol % to about 50 mol % of the total lipid present in the composition. 
     
     
         86 . The composition of any one of  claims 1 to 85 , wherein at least a portion of the RNA and, if present, of one or more lipids, is present in particles, such as lipid nanoparticles (LNPs), liposomes, and/or lipoplexes (LPXs). 
     
     
         87 . The composition of  claim 86 , wherein the particles have a size of from about 30 nm to about 500 nm. 
     
     
         88 . The composition of any one of  claims 1 to 87 , wherein the RNA is mRNA or inhibitory RNA. 
     
     
         89 . The composition of any one of  claims 1 to 88 , wherein the RNA (i) comprises a modified nucleoside in place of uridine, wherein the modified nucleoside is preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U); (ii) has a coding sequence which is codon-optimized; and/or (iii) has a coding sequence whose G/C content is increased compared to the wild-type coding sequence. 
     
     
         90 . The composition of any one of  claims 1 to 89 , wherein the RNA comprises at least one of the following, preferably all of the following: a 5′ cap; a 5′ UTR; a 3′ UTR; and a poly-A sequence. 
     
     
         91 . The composition of  claim 90 , wherein the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence preferably is an interrupted sequence of A nucleotides. 
     
     
         92 . The composition of  claim 90 or 91 , wherein the 5′ cap is a cap1 or cap2 structure. 
     
     
         93 . The composition of any one of  claims 1 to 92 , wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and/or comprise an epitope for inducing an immune response against an antigen in a subject. 
     
     
         94 . The composition of  claim 93 , wherein the pharmaceutically active polypeptide and/or the antigen or epitope is derived from or is a protein of a pathogen, an immunogenic variant of the protein, or an immunogenic fragment of the protein or the immunogenic variant thereof. 
     
     
         95 . The composition of  claim 93 or 94 , wherein the pharmaceutically active polypeptide and/or the antigen or epitope is derived from or is a SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof. 
     
     
         96 . The composition of any one of  claims 1 to 95 , wherein the composition is in liquid form, preferably at a temperature of about 2° C. to about 10° C. 
     
     
         97 . The composition of any one of  claims 1 to 96 , wherein the RNA integrity of the composition after storage for at least one week, preferably at a temperature of about 2° C. to about 8° C., is at least 50% compared to the RNA integrity before storage. 
     
     
         98 . The composition of any one of  claims 87 to 97 , wherein the size (Z average ) and/or size distribution and/or polydispersity index (PDI) of RNA particles (in particular LNPs) after storage of the composition is essentially equal to the size (Z average ) and/or size distribution and/or PDI of the RNA particles before storage. 
     
     
         99 . The composition of any one of  claims 1 to 95 , wherein the composition is in frozen form. 
     
     
         100 . The composition of  claim 99 , wherein the RNA integrity after thawing the frozen composition is at least 50% compared to the RNA integrity before the composition has been frozen. 
     
     
         101 . The composition of  claim 99 or 100 , wherein the size (Z average ) and/or size distribution and/or polydispersity index (PDI) of RNA particles (in particular LNPs) after thawing the frozen composition is essentially equal to the size (Z average ) and/or size distribution and/or PDI of the RNA particles before the composition has been frozen. 
     
     
         102 . A method of preparing a composition comprising LNPs dispersed in a final aqueous phase, wherein the LNPs comprise a cationically ionizable lipid and RNA; the final aqueous phase comprises a final buffer system comprising a final buffer substance, the final buffer substance having the formula N(R 1 )(R 2 )(R 3 ), its N-oxide, or a protonated form thereof, wherein R 1 , R 2 , and R 3  are as defined in any one of  claims 1 to 38 ;
 wherein the method comprises:   (I) preparing a formulation comprising LNPs dispersed in the final aqueous phase, wherein the LNPs comprise the cationically ionizable lipid and RNA; and   (II) optionally freezing the formulation to about −10° C. or below,   thereby obtaining the composition,   wherein step (I) comprises:   (a) preparing an RNA solution containing water and a first buffer system;   (b) preparing an ethanolic solution comprising the cationically ionizable lipid and, if present, one or more additional lipids;   (c) mixing the RNA solution prepared under (a) with the ethanolic solution prepared under (b), thereby preparing a first intermediate formulation comprising the LNPs dispersed in a first aqueous phase comprising the first buffer system; and   (d) filtrating the first intermediate formulation prepared under (c) using a final aqueous buffer solution comprising the final buffer system,   thereby preparing the formulation comprising the LNPs dispersed in the final aqueous phase.   
     
     
         103 . The method of  claim 102 , wherein step (I) further comprises one or more steps selected from diluting and filtrating. 
     
     
         104 . The method of  claim 102 or 103 , wherein step (I) comprises:
 (a′) providing an aqueous RNA solution;   (b′) providing a first aqueous buffer solution comprising a first buffer system;   (c′) mixing the aqueous RNA solution provided under (a′) with the first aqueous buffer solution provided under (b′) thereby preparing an RNA solution containing water and the first buffer system;   (d′) preparing an ethanolic solution comprising the cationically ionizable lipid and, if present, one or more additional lipids;   (e′) mixing the RNA solution prepared under (c′) with the ethanolic solution prepared under (d′), thereby preparing a first intermediate formulation comprising LNPs dispersed in a first aqueous phase comprising the first buffer system;   (f′) optionally filtrating the first intermediate formulation prepared under (e′) using a further aqueous buffer solution comprising a further buffer system, thereby preparing a further intermediate formulation comprising the LNPs dispersed in a further aqueous phase comprising the further buffer system, wherein the further aqueous buffer solution may be identical to or different from the first aqueous buffer solution;   (g′) optionally repeating step (f′) once or two or more times, wherein the further intermediate formulation comprising the LNPs dispersed in the further aqueous phase comprising the further buffer system obtained after step (f′) of one cycle is used as the first intermediate formulation of the next cycle, wherein in each cycle the further aqueous buffer solution may be identical to or different from the first aqueous buffer solution;   (h′) filtrating the first intermediate formulation obtained in step (e′), if step (f′) is absent, or the further intermediate formulation obtained in step (f′), if step (f′) is present and step (g′) is not present, or the further intermediate formulation obtained after step (g′), if steps (f′) and (g′) are present, using a final aqueous buffer solution comprising the final buffer system; and   (i′) optionally diluting the formulation obtained in step (h′) with a dilution solution;   thereby preparing the formulation comprising the LNPs dispersed in the final aqueous phase.   
     
     
         105 . The method of any one of  claims 102 to 104 , wherein filtrating is tangential flow filtrating or diafiltrating, preferably tangential flow filtrating. 
     
     
         106 . The method of any one of  claims 102 to 105 , which comprises (II) freezing the formulation to about −10° C. or below. 
     
     
         107 . The method of  claim 106 , wherein the formulation obtained in step (I) and the composition comprise a cryoprotectant. 
     
     
         108 . The method of any one of  claims 102 to 107 , wherein the final buffer substance is selected from BTM and its protonated form, TEA and its protonated form, ethyldiethanolamine and its protonated form, 2-(diethylamino)ethan-1-ol and its protonated form, triethylamine and its protonated form, 2-[2-(diethylamino)ethoxy]ethan-1-ol and its protonated form, diethanolamine and its protonated form, N,N′-bis(2-hydroxyethyl)piperazine and its protonated form, N,N,N′,N′-tetrakis(2-hydroxyethyl)ethylenediamine and its protonated form, and trimethylamine N-oxide and its protonated form. 
     
     
         109 . The method of any one of  claims 102 to 108 , wherein the concentration of the final buffer substance in the composition is between about 10 mM to about 200 mM, preferably between about 15 mM to about 100 mM, more preferably between about 20 mM to about 80 mM, more preferably between about 40 mM to about 60 mM, such as about 50 mM. 
     
     
         110 . The method of any one of  claims 102 to 109 , wherein (i) the RNA solution obtained in step (a) has a pH of below 6.0, preferably at most about 5.0, more preferably at most about 4.5; or (ii) the first aqueous buffer solution has a pH of below 6.0, preferably at most about 5.0, more preferably at most about 4.5. 
     
     
         111 . The method of any one of  claims 102 to 110 , wherein (i) the first buffer system used in step (a) comprises the final buffer substance used in step (d), preferably the buffer system and pH of the first buffer system used in step (a) are identical to the buffer system and pH of the final aqueous buffer solution used in step (d); or (ii) each of the first buffer system and every further buffer system used in steps (b′), (f′) and (g′) comprises the final buffer substance used in step (h′), preferably the buffer system and pH of each of the first aqueous buffer solution and of every further aqueous buffer solution used in steps (b′), (f′) and (g′) are identical to the buffer system and pH of the final aqueous buffer solution. 
     
     
         112 . The method of any one of  claims 102 to 111 , wherein the pH of the composition is between about 4.0 and about 8.0, preferably between about 4.5 and about 8.0, such as between about 5.0 and about 8.0, between about 5.5 and about 8.0, between about 6.0 and about 8.0, between about 6.5 and about 8.0, between about 6.8 and about 7.9, or between about 7.0 and about 7.8. 
     
     
         113 . The method of any one of  claims 102 to 112 , wherein water is the main component in the formulation and/or composition and/or the total amount of solvent(s) other than water contained in the composition is less than about 0.5% (v/v). 
     
     
         114 . The method of any one of  claims 102 to 113 , wherein the osmolality of the composition is at most about 1000×10 −3  osmol/kg, preferably between about 100×10 −3  osmol/kg and about 750×10 −3  osmol/kg, such as between about 100×10 −3  osmol/kg and about 500×10 −3  osmol/kg, more preferably about 300×10 −3  osmol/kg. 
     
     
         115 . The method of any one of  claims 102 to 114 , wherein the concentration of the RNA in the composition is about 5 mg/l to about 500 mg/l, such as about 10 mg/l to about 400 mg/l, about 10 mg/l to about 300 mg/l, about 10 mg/l to about 200 mg/l, about 10 mg/l to about 150 mg/l, or about 10 mg/l to about 100 mg/l, preferably about 10 mg/l to about 140 mg/l, more preferably about 20 mg/l to about 130 mg/l, more preferably about 30 mg/l to about 120 mg/l. 
     
     
         116 . The method of any one of  claims 102 to 115 , wherein the cationically ionizable lipid comprises a head group which includes at least one nitrogen atom which is capable of being protonated under physiological conditions. 
     
     
         117 . The method of any one of  claims 102 to 116 , wherein the ethanolic solution prepared in step (b) or (d′) further comprises one or more additional lipids and the LNPs further comprise the one or more additional lipids, wherein the one or more additional lipids are preferably selected from the group consisting of polymer conjugated lipids, neutral lipids, steroids, and combinations thereof, more preferably the one or more additional lipids comprise a polymer conjugated lipid, a neutral lipid (e.g., a phospholipid), and a steroid. 
     
     
         118 . The method of any one of  claims 102 to 117 , wherein the cationically ionizable lipid, the polymer conjugated lipid, the neutral lipid, and the steroid are present in the ethanolic solution in a molar ratio of 20% to 60% of the cationically ionizable lipid, 0.5% to 15% of the polymer conjugated lipid, 5% to 25% of the neutral lipid (e.g., phospholipid), and 25% to 55% of the steroid, preferably in a molar ratio of 45% to 55% of the cationically ionizable lipid, 1.0% to 5% of the polymer conjugated lipid, 8% to 12% of the neutral lipid, and 35% to 45% of the steroid. 
     
     
         119 . The method of any one of  claims 102 to 118 , wherein the RNA is as defined in any one of  claims 88 and 89 to 95 . 
     
     
         120 . The method of any one of  claims 102 to 105 and 107 to 119 , which does not comprise step (II). 
     
     
         121 . A method of preparing an aqueous RNA composition, wherein the method comprises:
 (I) preparing a formulation comprising RNA and an aqueous phase, wherein the aqueous phase comprises a buffer substance, the buffer substance having the formula N(R 1 )(R 2 )(R 3 ), its N-oxide, or a protonated form thereof, wherein R 1 , R 2 , and R 3  are as defined in any one of  claims 1 to 38 ; and   (II) optionally freezing the formulation to about −10° C. or below,   thereby obtaining the composition.   
     
     
         122 . The method of  claim 121 , which comprises (II) freezing the formulation to about −10° C. or below. 
     
     
         123 . The method of  claim 122 , wherein the composition comprises a cryoprotectant. 
     
     
         124 . The method of  claim 121 , which does not comprise step (II). 
     
     
         125 . The method of any one of  claims 121 to 124 , wherein the buffer substance is selected from BTM and its protonated form, TEA and its protonated form, ethyldiethanolamine and its protonated form, 2-(diethylamino)ethan-1-ol and its protonated form, triethylamine and its protonated form, 2-[2-(diethylamino)ethoxy]ethan-1-ol and its protonated form, diethanolamine and its protonated form, N,N′-bis(2-hydroxyethyl)piperazine and its protonated form, N,N,N′,N′-tetrakis(2-hydroxyethyl)ethylenediamine and its protonated form, and trimethylamine N-oxide and its protonated form. 
     
     
         126 . A method of storing a composition, comprising preparing a composition according to the method of any one of  claims 102 to 119, 121, 122, and 125  and storing the composition at a temperature ranging from about −90° C. to about −10° C., such as from about −90° C. to about −40° C. or from about −25° C. to about −10° C. 
     
     
         127 . The method of 126, wherein the composition comprises a cryoprotectant. 
     
     
         128 . The method of  claim 127 , wherein storing the composition is for at least 1 month, such as at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months. 
     
     
         129 . A method of storing a composition, comprising preparing a composition according to the method of any one of  claims 102 to 128  and storing the composition at a temperature ranging from about 0° C. to about 20° C., such as from about 1° C. to about 15° C., from about 2° C. to about 10° C., or from about 2° C. to about 8° C., or at a temperature of about 5° C. 
     
     
         130 . The method of  claim 129 , wherein storing the composition is for at least 1 week, such as at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, or at least 24 months. 
     
     
         131 . A composition preparable by the method of any one of  claims 102 to 130 . 
     
     
         132 . The composition of  claim 131 , which is in frozen form. 
     
     
         133 . The composition of  claim 132 , further comprising a cryoprotectant. 
     
     
         134 . The composition of  claim 132 or 133 , wherein the RNA integrity after thawing the frozen composition is at least 50% compared to the RNA integrity of the composition before the composition has been frozen. 
     
     
         135 . The composition of any one of  claims 132 to 134 , wherein the size (Z average ) and/or size distribution and/or polydispersity index (PDI) of RNA particles after thawing the frozen composition is essentially equal to the size (Z average ) and/or size distribution and/or PDI of the RNA particles before the composition has been frozen. 
     
     
         136 . The composition of  claim 131 , which is in liquid form. 
     
     
         137 . The composition of  claim 136 , wherein the RNA integrity after storage of the composition for at least 1 week is at least 50% compared to the RNA integrity before storage. 
     
     
         138 . The composition of  claim 136 or 137 , wherein the size (Z average ) and/or size distribution and/or polydispersity index (PDI) of RNA particles after storage of the composition for at least one week is essentially equal to the size (Z average ) and/or size distribution and/or PDI of the RNA particles before storage. 
     
     
         139 . A method for preparing a ready-to-use pharmaceutical composition, the method comprising the steps of providing a frozen composition prepared by the method of any one of  claims 102 to 119, 121, 122, 123, and 125 to 128 , and thawing the frozen composition thereby obtaining the ready-to-use pharmaceutical composition. 
     
     
         140 . A method for preparing a ready-to-use pharmaceutical composition, the method comprising the step of providing a liquid composition prepared by the method of any one of  claims 102 to 105, 107 to 121, 124, 125, 129, and 130 , thereby obtaining the ready-to-use pharmaceutical composition. 
     
     
         141 . A ready-to-use pharmaceutical composition preparable by the method of  claim 139 or 140 . 
     
     
         142 . A composition of any one of  claims 1 to 101, 131 to 138, and 141  for use in therapy. 
     
     
         143 . A composition of any one of  claims 1 to 101, 131 to 138, and 141  for use in inducing an immune response in a subject.